1. Field of the Invention
This invention relates to a gene and an adhesion molecule encoded by said gene, and in particular, relates to a gene encoding a polypeptide having pre-B cell growth-supporting ability, a vector containing said gene, transformants such as microorganisms or cells transformed by said vector and a method for producing the adhesion molecule having pre-B cell growth-supporting ability by using said gene.
The gene of the present invention encodes a novel adhesion molecule enhancing pre-B cell growth-supporting ability on the surface of bone marrow cells and synovial cells derived from patients with rheumatoid arthritis (RA) or multiple myeloma (MM). In the present invention, a homogeneous and purified adhesion molecule having pre-B cell growth-supporting ability can be produced in large quantities by transforming appropriate host cells with a suitable vector in which the gene of the present invention is inserted. Thus, according to the present invention, it becomes possible to identify multiple myeloma (MM) and rheumatoid arthritis (RA), and also prepare reagents for the clinical diagnosis thereof.
2. Description of the Prior Art
It has been reported that abnormalities of bone marrow cells are actually involved in the pathogenesis of B cell malignancies and autoimmune disease [Annu. Rev. Immunol., 9:243 (1991)].
Namely, multiple myeloma (MM) is a tumor which develops depending on microenvironment in the bone marrow and also a monoclonal plasmacytic tumor characterized by restricted growth in the bone marrow, and several studies have suggested that the oncogenic transformation of multiple myeloma (MM) occurred during the process of the differentiation and proliferation of early B cell development (pre-B cell) which is dependent on bone marrow stromal cells [J. Exp. Med., 150: 792 (1979), and Cancer Genet. Cytogenet, 17:13 (1985)].
Furthermore, the fact has been reported that bone marrow stromal cells were demonstrated to induce the growth of the precursor cells of multiple myeloma (MM) circulating in the peripheral blood of patients with multiple myeloma (MM) [Blood, 77:2688 (1991)].
Therefore, it is probable that bone marrow stromal cells provide stimulatory signals essential for the generation of multiple myeloma (MM).
In this regard, it has been known that the abnormal production of IL-6 may play a role in pathogenesis of rheumatoid arthritis (RA) [Eur. J. Immunol., 18:1797 (1988), and Clin. Immunol. Immunopathol., 62:S60 (1992)]. Besides, according to the results taking several murine autoimmune models into consideration reported in Eur. J. Immunol., 20:723 (1990) and Eur. J. Immunol., 21:63 (1991), it is suggested that the bone marrow derived from patients with rheumatoid arthritis (RA) is probably affected.
Namely, also in rheumatoid arthritis (RA), it is suggested that polyclonal B cell activation is probably caused by the bone marrow adjacent to an affected arthrosis.
The present inventors have engaged in extensive studies with a view to investigate the function of the bone marrow microenvironments in disorders causing abnormalities of B cells, and have reported that the pre-B cell growth-supporting ability of BM stromal cells derived from patients with RA or MM is enhanced in comparison with that of healthy donor-derived BM stromal cells, and that the direct cell-to-cell interaction of pre-B cells and stromal cells might play essential roles in this supporting ability. At the same time, the present inventors have obtained bone marrow specimens derived from patients with MM and RA after informed consent, and established novel stromal cell lines (RASV5-5, MMSV3-3) containing a molecule enhancing the growth of pre-B cells, on the basis of the speculation that there should be a surface molecule enhancing the growth of pre-B cells on the bone marrow stromal cells derived from patients with RA and MM. It has been suggested that the pre-B cell growth-supporting activity of these stromal cell lines is most likely caused by unknown adhesion molecules different from known Stem cell factors (SCF), ICAM-1, CD44, VCAM-1, LFA-1xcex1, LFA-1xcex2, NCAM and ELAM-1 [J. Immunol., 149:4088 (1992)].
Therefore, the present inventors have prepared for a monoclonal antibody using the BM stromal cell line RASV5-5 having enhanced pre-B cell growth-supporting ability derived from patients with RA as an antigen for immunization, and obtained a monoclonal antibody RF3 responding to BM stromal cell lines derived from patients with RA and MM, and not responding to a stromal cell line NFSV1-1 derived from the human bone marrow having no pre-B cell growth-supporting ability.
Further, the present inventors have found that a monoclonal antibody yielded by the hybridoma SG2, which obtained from the immunized cell line SynSV6-14 of the synovial cell derived from patients with RA, responds to BM stromal cell line RASV5-5 derived from patients with RA, and does not respond to the stromal cell line NFSV1-1 derived from the human bone marrow having no pre-B cell growth-supporting ability.
This invention is directed to a gene and an adhesion molecule encoded by said gene, and in particular, relates to a gene encoding a polypeptide having pre-B cell growth-supporting ability, a vector containing said gene, transformants such as microorganisms or cells transformed by said vector and a method for producing the adhesion molecule having pre-B cell growth-supporting ability by using said gene.
The present invention provides a gene encoding the polypeptide having human pre-B cell growth-supporting ability.
The present invention also provides a recombinant vector containing the gene encoding the polypeptide having human pre-B cell growth-supporting ability.
The present invention further provides a procaryotic or eucaryotic host cell transformed by the recombinant vector containing the gene encoding the polypeptide having human pre-B cell growth-supporting ability.
Furthermore, the present invention provides a method for producing the polypeptide having human pre-B cell growth-supporting ability characterized by culturing a transformant such as microorganisms or cells transformed by the recombinant vector containing the gene encoding the polypeptide having human pre-B cell growth-supporting ability.
The gene of the present invention encodes a novel adhesion molecule enhancing pre-B cell growth-supporting ability on the surface of bone marrow cells and synovial cells derived from patients with rheumatoid arthritis (RA) or multiple myeloma (MM). In the present invention, a homogeneous and purified adhesion molecule having pre-B cell growth-supporting ability can be produced in large quantities by transforming appropriate host cells with a suitable vector in which the gene of the present invention is inserted. Thus, according to the present invention, it becomes possible to identify multiple myeloma (MM) and rheumatoid arthritis (RA), and also prepare reagents for the clinical diagnosis thereof.
Since it is probable that the expression of a novel adhesion molecule having pre-B cell growth-supporting ability recognized by the above-mentioned hybridomas is probably correlate with the degree of the disorders of patients with RA and MM, it becomes to be important problems that investigating the properties of the membrane protein, and securing thereof in large quantities capable of being utilized for diagnosis, remedies and studies of RA and MM. Therefore, investigating the structures of genes of the adhesion molecule and establishing the mass production technique of the adhesion molecule by using recombination DNA technique have been desired.
That is, the present invention is directed to provide a gene encoding a polypeptide having pre-B cell growth-supporting ability, a vector containing said gene, transformants such as microorganisms or cells transformed by said vector and a method for producing the adhesion molecule having pre-B cell growth-supporting ability by using said gene.
Under such circumstances, the present inventors have prepared cDNAs library from mRNA prepared from cells expressing an adhesion molecule, and then inserted these cDNAs into an expression vector to obtain transformants such as microorganisms or cells transformed by the expression vector. Subsequently, the present inventors have succeeded in cloning the genes of a novel adhesion molecule having pre-B cell growth-supporting ability by repeating the steps, which consist of selecting transformants responding strongly to a monoclonal antibody produced by said hybridomas, further sorting cDNAs used for the preparation thereof and inserting them into the expression vector again, selecting transformants responding strongly to said antibody, and further sorting cDNAs used for the preparation thereof. In addition, the present inventors have found that the novel adhesion molecule can be produced in large quantities, by obtaining a transformant by inserting the gene into a proper vector, and then culturing it.
Therefore, the present invention provides a gene encoding the polypeptide having human pre-B cell growth-supporting ability.
The present invention also provides a recombinant vector containing the gene encoding the polypeptide having human pre-B cell growth-supporting ability.
The present invention further provides a procaryotic or eucaryotic host cell transformed by the recombinant vector containing the gene encoding the polypeptide having human pre-B cell growth-supporting ability.
Furthermore, the present invention provides a method for producing the polypeptide having human pre-B cell growth-supporting ability characterized by culturing a transformant such as microorganisms or cells transformed by the recombinant vector containing the gene encoding the polypeptide having human pre-B cell growth-supporting ability.
Subsequently, the present invention will be described in detail.
The gene of the present invention is obtained, for example, by preparing mRNA from a cell expressing an adhesion molecule having human pre-B cell growth-supporting ability, and then converting it into a double-stranded cDNA according to a known method. As a cell used for preparing the mRNA can be mentioned cell lines RASV5-5 and SynSV6-14 used as immune sources of hybridomas RF3 and SG2, but it is not limited to these cell lines and therefore any type of cells expressing the adhesion molecule having human pre-B cell growth-supporting ability may be used. As one example thereof can be mentioned various stromal cell lines disclosed in J. Immunol., 149:4088 (1992). Incidentally, SynSV1-4 was used in the present invention.
For the preparation of the total RNA for obtaining mRNA can be employed a method for obtaining the total RNA which consists of performing cesium chloride density-gradient centrifugation after a guanidine thiocyanate treatment [Chirgwin et al., Biochemistry, 18:5294 (1979)], a method which consists of performing a surfactant treatment and a phenol treatment in the presence of the ribonuclease inhibitor of a vanadium complex [Berger and Birkenmeier, Biochemistry, 18:5143 (1979)], and other known methods.
The preparation of mRNA from the total RNA, can be accomplished by recovering poly(A) +RNA from the total RNA according to, for example, affinity column chromatography using Sephalose or cellulose, or a batch method. Besides, poly(A) +RNA can be further purified according to sucrose density-gradient centrifugation. In addition, there can be mentioned a method for obtaining poly(A) +RNA directly without preparing RNA or a convenient method using a commercially available kit.
In order to obtain a double-stranded cDNA from the thus obtained mRNA, for example, a DNA (cDNA) complementary to mRNA is synthesized by using mRNA as a template, and using an oligo (dT) complementary to a poly-A-chain sited at the 3xe2x80x2 end as a primer, and the treating it with reverse transcriptase.
The double-stranded cDNA can be also obtained by degrading mRNA according to an alkaline treatment, subjecting the obtained single-stranded cDNA as a template to a treatment with reverse transcriptase or DNA polymerase (e.g., Klenow fragment), and then treating it with S1 nuclease, or treating it directly with RNase and DNA polymerase [Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory (1982) and Gubler and Hoffman, Gene, 25:263 (1983)]. Nowadays, convenient kits have been on the market, and a double-stranded cDNA can be obtained by using them.
The cDNAs library can be obtained by inserting the thus obtained cDNA into a proper vector, for example, an EK-type plasmid vector such as pBR322 and pSC101, and a phage vector such as xcexgt10, and then transforming Escherichia coli with said vector (e.g., X1776, HB101, DH1, DH5) or the like (refer, for example, to xe2x80x9cMolecular Cloningxe2x80x9d above).
On the other hand, host cells of other procaryotes and eucaryotes can be transformed by using a suitable expression vector in which the double-stranded cDNA obtained according to the above-mentioned method is inserted.
The ligation of the double-stranded cDNA to the vector can be performed by adding a proper chemically-synthesized DNA adapter thereto, and subjecting it with a vector DNA cleavaged by means of a restriction enzyme to a treatment with T4 phage DNA ligase in the presence of ATP.
The expression vector of the present invention contains a replicative origin, a selective marker, a promoter located in upstream region of a gene to be expressed, an RNA splice site and a polyadenylated signal.
As a gene expression promoter in a mammal cell may be used virus promoters such as retrovirus, polyoma virus, adenovirus and simian virus (SV) 40, and promoters derived from cells such as human polypeptide chain elongation factor 1xcex1 (HEF-1xcex1). For example, in case of using a promoter of SV40, it can be performed easily according to a method of Mulligan et al. [Nature, 277:108 (1979)].
As a replicative origin can be used those derived from SV40 polyoma virus, adenovirus and bovine papilloma virus (BPV), and as a selective marker can be used a phosphotransferase APH (3xe2x80x2) II or I (neo) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene and a dihydrofolate reductase (DHFR) gene.
In order to express the desired gene using a procaryotic cell as a host cell, the host cell is transformed with a replicon derived from species capable of being fitted as hosts, namely, a plasmid vector containing a replicative origin and a regulation sequence. A vector which has a marker gene capable of imparting the selectivity of a phenotype to transformed cells is preferable. For example, in case of using Escherichia coli as a host cell, it can be transformed using pBR322, a vector originated from the host cell [Boliver et al., Gene, 2:95 (1975)]. The pBR322 contains an ampicillin resistant gene and a tetracycline resistant gene, and therefore transformants can be identified by utilizing either of these resistant properties. As a promoter needed for the gene expression of a procaryotic host cell can be mentioned a promoter of a xcex2-lactamase gene [Chang et al., Nature, 275:615 (1978)], a lactose promoter [Goeddle et al., Nature, 281:544 (1979)], a tryptophan promoter [Goeddle et al., Nucleic Acid Res., 8:4057 (1980)], a tac promoter and the like.
As procaryotic host cells of hosts to be used in the expression system of the present invention can be mentioned Escherichia coli, Bacillus subtilis, Bacillus thermophilus and the like.
In addition, as eucaryotic host cells can be mentioned eucaryotic microorganisms such as Saccharomyces cerevisiae, and cells derived from mammals such as a COS cell, a Chinese hamster ovary (CHO) cell, a C127 cell, a 3T3 cell, a Hela cell, a BHK cell, a namalwa cell and a human fetal renal cell (293 cell).
Incidentally, the culture of the transformants of the present invention may be performed by selecting culture conditions suitable for host cells appropriately,
The isolation of a cDNA encoding an adhesion molecule having pre-B cell growth-supporting ability can be performed, for example, by using pre-B cell growth-supporting ability as an index or according to a method such as direct expression cloning using an antibody. The measurement of pre-B cell growth-supporting ability can be performed by using a murine pre-B cell line DW34 [Eur. J. Immunol., 18:1767 (1988)].
That is, a cell expressing the adhesion molecule having pre-B cell growth-supporting ability is cultured until it becomes subconfluent on 24-well plates (preferable density being about 50%) and a proper amount of radiation is irradiated thereupon, DW34 of 1 to 2xc3x97103 per well is added thereto, and cultured in RPMI-1640 medium containing 10% FCS under the condition of 5% CO2 at 37xc2x0 C. for about 4 to 6 days. The degree of the enhancement of the growth-supporting ability can be found by examining the number of living cells of DW34 in each well according to trypan blue dye exclusion.
In the present invention, the desired gene could be cloned by repeating the steps, which consists of selecting a transformant expressing an adhesion molecule according to an FACScan using monoclonal antibodies RF3 and SG2 recognizing the adhesion molecule having pre-B cell growth-supporting ability, preparing a transformant again by sorting the plasmid DNA used for the preparation of the transformant, and then screening the transformant according to flow cytometry.
Namely, a transduced transformant (293T cell) was cultured on well plates and removed from the plates with PBS containing 0.02% EDTA, and after the cell was washed with an FACS buffer solution composed of PBS containing 2% FCS and 0.02% NaN3, it was reacted with RF3 and SG2 as primary antibodies. Subsequently, after the unreacted primary antibodies were removed by washing it with an FACS buffer solution, it was further reacted with a secondary antibody, an FITC-labeled antibody (FITC-labeled anti-mouse goat Ig antibody), dead cells were stained with propidium iodide, and viable cells were analyzed by an FACScan to select transformants responding strongly to RF3 and SG2.
Further, the complete length of cDNA (63-BOS) encoding a membrane protein polypeptide having novel pre-B cell growth-supporting ability shown in sequence No. (SEQ ID NO) 2 of the sequence table (SEQUENCE TABLE) could be obtained by repeating the steps, which consists of treating Escherichia coli (DH5) containing the cDNA used for the preparation of transformants responding to antibodies with alkali to select a group of plasmids containing the desired gene, subdividing the group of plasmids into some groups of plasmids, transducing them into 293T cells again, and then selecting transformants according to FACScan analysis using the above-mentioned monoclonal antibodies RF3 and SG2.
Incidentally, the Escherichia coli DH5xcex1 strain containing pBst-1 with the cDNA inserted into the XbaI cleavage sites of a pUC19 vector was deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology in Japan (Address: 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305, JAPAN), which is international depositary authority according to Budapest Treaty on the international recognition of the deposit of microorganisms for the purpose of patent procedure, on May 19, 1993, under the name of Escherichia Coli DH5 xcex1 (pBst-1) with accession No. FERM BP-4305.
Generally, the genes of eucaryotes are thought to show polymorphism as known according to human interferon genes [e.g., Nishi et al., J. Biochem., 97: 153 (1985)], and in some cases at least one amino acid is substituted according to this polymorphism, and in other cases amino acids do not change at all though there are changes in the DNA sequence.
Further, it is probable that some polypeptides having at least one more or less amino acid than the amino acid sequence shown in sequence No. 1 of the sequence table, or some polypeptides substituted with at least one amino acid may have also pre-B cell growth-supporting ability. Actually, for example, it has been already known that the polypeptide obtained from a human interleukin (IL-2) gene, in which a DNA sequence corresponding to cysteine is converted to a sequence corresponding to serine, holds also an IL-2 activity [Wang et al., Science, 224:1431 (1984)].
Moreover, a known protein gene and a gene shown in sequence No. 2 of the sequence table can be ligated by means of a proper restriction enzyme or adaptor to yield a polypeptide bound to the known protein. As this know protein gene can be mentioned immunoglobulin, and it may be bound to an Fc portion of it using the gene shown in sequence No. 2 of the sequence table instead of the variable region site of it [(Zettlmeissl et al., DNA AND CELL BIOLOGY, 9:347-353 (1990)].
Furthermore, in case of expressing a polypeptide in eucaryotic cells, glycosylation occurs in many cases, and the glycosylation can be regulated according to the conversion of at least one amino acid; in this case, too, they may have pre-B cell growth-supporting ability.
Therefore, even the genes in which the site encoding a polypeptide having pre-B cell growth-supporting ability are modified artificially can be included in the present invention so far as the polypeptides obtained from the genes have pre-B cell growth-supporting ability.
Moreover, genes to be hybridized with genes shown in sequence No. 2 of the sequence table are also included in the present invention so far as the polypeptides expressed from the genes have pre-B cell growth-supporting ability. Hybridization may be carried out according to employing ordinary hybridization conditions (for example, refer to the above-mentioned xe2x80x9cMolecular Cloningxe2x80x9d).
A homogeneous and purified soluble adhesion molecule can be obtained by culturing a transformant such as microorganisms or cells transformed with a gene encoding a polypeptide having pre-B cell growth-supporting supporting ability to be desired, solubilizing the yielded polypeptide with proper detergent, subjecting the resultant polypeptide to separation and purification. Examples of the detergent include Nonidet P-40 (NP-40), Sodium Dodecyl Sulphate (SDS), Triton X-100, Tween 20 and the like.
In addition, the soluble adhesion molecules can be also prepared according to gene engineering. Namely, since the portion from the 272nd Gln to the 290th Leu in sequence No. 1 of the sequence table is a region with high hydrophobic properties, a gene having a termination codon at a position before the 272nd position can be prepared by employing a PCR-mutagenesis method [M. Kamman et al., Nucl. Acids Res., 15:5404 (1989)].
Also describing in detail, the gene of the deposited pBst-1 is amplified according to the PCR-mutagenesis method employing two primers AAC CTC CAG AAG GAA AA (corresponding to the 185th to the 190th of sequence No. 1 of the sequence table) and ACC CAA GCT TTC TAG ATC AAT AAA GAC TTG GGG CTT (corresponding to the 264th to the 269th of sequence No. 1 of the sequence table + a termination codon + HindIII and the XbaI restriction site). The desired gene is purified using low melting point agarose and the like, and cleaved to fragments with BglII and HindIII. After the obtained BglII-HindIII fragment is inserted into the pBst-1 which was digested with the same restriction enzyme, it is digested with EcoRI and HindIII, and treated with a Klenow fragment to make a flush end. The DNA fragment is ligated with pEF-BOS which is digested with BstXI, and treated with the Klenow fragment, and a proper host cell is transformed with it to yield a soluble adhesion molecule.
As a means of separation and purification of the molecule, a method to be used in the case of ordinary protein can be employed; for example, the polypeptide of the present invention can be separated and purified properly by selecting and combining various types of chromatography such as affinity chromatography using the above-mentioned monoclonal antibodies, ultrafiltration, salting out, dialysis and the like.